The therapeutic and preventive application of probiotics for necrotizing enterocolitis (NEC) has been supported by more and more experimental and clinical evidence in which Toll-like receptor 4 (TLR4) exerts a significant role. In immune cells, probiotics not only regulate the expression of TLR4 but also use the TLR4 to modulate the immune response. Do probiotics also use the TLR4 in immature enterocytes for anti-inflammation? Here we demonstrate that probiotic conditioned media (PCM) from Bifidobacterium longum supp infantis but not isolated organisms attenuates interleukin-6 (IL-6) induction in response to IL-1β by using TLR4 in a human fetal small intestinal epithelial cell line (H4 cells), human fetal small intestinal xenografts, mouse fetal small intestinal organ culture tissues and primary NEC enterocytes. Furthermore, we show that PCM, using TLR4, down regulates the mRNA expression of interleukin -1 receptor - associated kinase 2 (IRAK2), a common adapter protein shared by IL-1β and TLR4 signaling. PCM also reduces the phosphorylation of the activator-protein 1(AP-1) transcription factors c-Jun and c-Fos in response to IL-1β stimulation in a TLR4-dependent manner. This study suggests that PCM may use TLR4 through IRAK2, and via AP-1 to prevent IL-1β-induced IL-6 induction in immature enterocytes. Based on these observations, the combined use of probiotics and anti-TLR4 therapy to prevent NEC may not be a good strategy.